Antibacterial Activity of Ulva/Nanocellulose and Ulva/Ag/Cellulose Nanocomposites and Both Blended with Fluoride against Bacteria Causing Dental Decay

One of the most prevalent chronic infectious disorders is tooth decay. Acids produced when plaque bacteria break down sugar in the mouth cause tooth decay. Streptococcus mutans and Lactobacillus acidophilus are the most prominent species related to dental caries. Innovative biocidal agents that integrate with a biomaterial to prevent bacterial colonization have shown remarkable promise as a result of the rapid advancement of nanoscience and nanotechnology. In this study, Ulva lactuca was used as a cellulose source and reducing agent to synthesize nanocellulose and Ulva/Ag/cellulose/nanocomposites. The characterizations of nanocellulose and Ulva/Ag/cellulose/nanocomposites were tested for FT-IR, TEM, SEM, EDS, XRD, and zeta potential. Ulva/Ag/cellulose/nanocomposites and Ulva/nanocellulose, both blended with fluoride, were tested as an antibacterial against S. mutans ATCC 25175 and L. acidophilus CH-2. The results of the SEM proved that nanocellulose is filament-shaped, and FT-IR proved that the functional groups of Ulva/nanocellulose and Ulva/Ag/cellulose/nanocomposites and cellulose are relatively similar but present some small diffusion in peaks. The TEM image demonstrated that the more piratical size distribution of Ulva/Ag/cellulose/nanocomposites ranged from 15 to 20 nm, and Ulva/nanocellulose ranged from 10 to 15 nm. Ulva/Ag/cellulose/nanocomposites have higher negativity than Ulva/nanocellulose. Ulva/Ag/cellulose/nanocomposites and Ulva/nanocellulose possess antibacterial activity against S. mutans ATCC 25175 and L. acidophilus CH-2, but Ulva/Ag/cellulose/nanocomposites are more effective, followed by that blended with fluoride. It is possible to use Ulva/Ag/cellulose/nanocomposites as an antimicrobial agent when added to toothpaste. It is promising to discover an economic and safe nanocomposite product from a natural source with an antimicrobial agent that might be used against tooth bacteria.


Introduction
One of the most significant problems in public health is tooth decay [1]. General health is affected by oral health [2]. Dental caries is related to behavioral, economic, and social aspects and increases the incidence of diseases among people [3]. Dental caries is a bacterial illness that gradually arises from complicated biological interactions between acidogenic bacteria and fermentable carbohydrates [4]. Acids from bacterial metabolism that diffuse into enamel and dentine generate the bacterial disease process known as dental caries, which is contagious [5]. Worldwide, 36% of people suffer dental caries in their adult teeth (Gram-positive bacteria creating tooth decay) [6]. Dental caries is caused by The materials used in the study were green alga (Ulva lactuca), sodium hydroxide (NaOH), ethanol (99%), hydrochloric acid (37%), hydrogen peroxide (6%), silver nitrate 99.9+% (metals basis), and distilled water. All the chemicals used in this research were of analytical grade and applied without further purification. Chemical materials were purchased from the Saudi Chemical company (PanReac AppliChem, Ar Riyad, Saudi Arabia).

Alga Collection
Alga was collected from the Red Sea shore in Jeddah, Saudi Arabia (21 • 38 43.4 N 39 • 06 04.7 E). To remove impurities, alga was rinsed in water and then dried in an oven set to 60 • C. Crushed and sieved dry samples were used.

Extracting Cellulose from U. lactuca Green Alga
Ulva lactuca (50 g) was milled to a soft powder. After that, 50 g of grounded alga was placed in a flask with 170 mL of pure ethanol and 30 mL of water for 6 h over heat at 60 • C with a magnet (stirring) and filtered, and the liquid phase was discarded. The insoluble fraction was repeatedly cleaned with 99% ethanol before being dried for 16 h at 37 • C in the oven. After drying, the sample was further processed by suspending it in 400 mL of 4% H 2 O 2 , which was then heated to 80 • C for 16 h to remove any remaining green pigments and other colored impurities. The mixture was filtered, and the liquid phase was discarded after cooling to room temperature. The insoluble fraction was then suspended in 400 mL of 0.5 M Na OH after being rinsed with distilled water. For 16 h, the mixture was maintained at 60 • C and was then taken out of the oven, allowed to cool to room temperature, filtered, and washed three times with distilled water before the insoluble fraction was collected.

Extraction of Nanocellulose
The extracted cellulose was treated with 27 mL HCl and 173 mL of distilled water at 90 • C for 10 min. The combination was filtered by removing the liquid phase after cooling to room temperature, and the insoluble fraction was then cleaned with distilled water. When the mixture was entirely dry, it was stored at 60 • C.

Biosynthesis of Ag Nanoparticles
Silver nitrate (0.17 g) was added to 90 mL of distilled water. The prepared alga extract (10 mL) was added drop-wise to the solution at 60 • C with constant stirring until the mixture turned brown.

Biosynthesis of Ulva/Ag/Cellulose Nanocomposites
Silver nitrate (0.085 g) was added to 45 mL of distilled water, then different concentrations of nanocellulose (0.1, 0.2, 0.4, and 0.8 g) were added separately, and then alga extract (5 mL) was added as drops. The solution was heated at 60 • C and stirred until the color changed to brown.
A field emission scanning electron microscope equipped with energy-dispersive spectroscopy (EDS) (JEOL JSM-6510/v, Tokyo, Japan) was used to investigate the surface morphology of the Ulva/nanocellulose and Ulva/Ag/cellulose nanocomposite.

Zeta Potential Analysis
The zeta potential of the Ulva/nanocellulose and Ulva/Ag/cellulose nanocomposite provides details of the stabilization in the middle of the liquid that it is dispersed in (Malvern Zeta size Nano-Zs90, Malvern, PA, USA).
2.6.7. Differential Scanning Calorimetry (DSC) Differential scanning calorimetry DSC testing 19 mg of Ulva/Ag/cellulose nanocomposite blend with fluoride was conducted using differential scanning calorimeter DSC131 EVO France.

Antibacterial Activities
The agar-well diffusion technique was used to study the antibacterial properties of hybrids made of AgNPs, Ulva/nanocellulose, and Ulva/Ag/cellulose nanocomposite, and both blended with fluoride against S. mutans ATCC 25175 and L. acidophilus CH-2 [38] as model Gram-positive bacteria associated with dental caries. Muller-Hinton agar was poured into a Petri dish and solidified. The turbidity of an overnight broth culture of S. mutans ATCC 25175 and L. acidophilus CH-2 was adjusted to 0.5 McFarland standards. Then, 50 µL of bacterial suspension was spread over the plates separately using a sterilized cotton swab. After that, 100 µL of the prepared nanoparticles at different concentrations of nanocellulose (0.2, 0.4, 0.8, and 1.6%) was applied to 0.7 mm-diameter wells on each bacterial agar plate. Plates were incubated at 37 • C for 24 h. The inhibitory zone (mm) was recorded. The experiment was conducted in triplicate.

Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) of Ulva/Ag/cellulose nanocomposite was examined using the standard broth dilution method at diverse concentrations oscillating from 0.002 to 0.00005 g/mL. Mueller-Hinton agar (MHA) medium was prepared and inoculated under aseptic conditions with 50 µL of the overnight bacteria suspension and allowed to dry. Wells were filled with 100 µL of different serial dilutions of Ulva/Ag/cellulose nanocomposite separately. After 24 h of incubation at 37 • C, plates were checked to see if an inhibition zone (mm) had formed around each well.

Statistical Analysis
Data were obtainable as mean ± SEM, and SPSS 16 was used for the statistics, together with one-way ANOVA.  Table 1 show the results of the FT-IR spectroscopy analysis of cellulose, nanocellulose, and Ulva/Ag/cellulose nanocomposite derived from U. lactuca. The results demonstrate that 9 peaks were obtained with cellulose, 18 peaks with nanocellulose, and 11 peaks with Ag/Ulva cellulose nanocomposites. The results investigated the modification of numbers and positions (wavenumbers) and demonstrated the differences in the chemical structure of each compound.

SEM and TEM Images
Figure 2a displays SEM (scanning electron microscope) images of the surface of nanocellulose extracted from U. lactuca. The image showed that nanocellulose extracted from U. lactuca comprises filaments, a result proved by Xiang et al. [63] who reported that the SEM imagery of Cladophora glomerata nanocellulose comprises filaments. Peng et al. [64] demonstrated that nanocellulose is arranged in a fiber network. The SEM image of Ulva/Ag cellulose nanocomposites indicates ridges and valley surfaces (Figure 2b), which confirmed a highly organized by layer porous architecture and large surface area. These results are confirmed by Tan [65], who reported that, after the addition of Ag rough, nonhomogeneous Turing structures increased. Figure 3a-b shows TEM images of biosynthesized nanocellulose and Ulva/Ag/cellulose nanocomposites derived from U. lactuca. The morphological studies of Ulva nanocellulose indicate a polydispersive and spherical shape and the major range size is from 10 to 15 nm (Figure 4a). The fine particle size will result in a large surface area that will enhance the nanoparticles' catalytic activity. The results appeared in TEM images of Ulva/Ag/cellulose nanocomposites that are polydispersed hexagonal-shaped nanoparticles with sizes ranging from 5 to 38.1 nm, however, the major size distribution was in the range of 15-20 nm (Figure 4b). The image clarifies that there is a shell core around the AgNPs, a core nanostructure that appears dark, in which nanocellulose appears as shells around the AgNPs. Capping of metal nanoparticles is one of the critical methods for ensuring its stability, AgNPs synthesized and capped by a secreted polysaccharideprotein matrix of Spirulina platensis were quasispherical shaped nanoparticles captured in a polysaccharide-protein matrix sheath with sizes ranging from 5.04 to 33.56 nm [66]. Rajeshkumar et al.'s [67] TEM results of AgNP-based chitosan nanocomposite found it to be spherical with a size of around 10-60 nm.
dark, in which nanocellulose appears as shells around the AgNPs. Capping of metal nanoparticles is one of the critical methods for ensuring its stability, AgNPs synthesized and capped by a secreted polysaccharide-protein matrix of Spirulina platensis were quasispherical shaped nanoparticles captured in a polysaccharide-protein matrix sheath with sizes ranging from 5.04 to 33.56 nm [66]. Rajeshkumar et al.'s [67] TEM results of AgNP-based chitosan nanocomposite found it to be spherical with a size of around 10-60 nm.

Energy-Dispersive X-ray Measurements
An analytical method called energy-dispersive X-ray spectroscopy can be used to determine the relative abundance of different elements in a given sample. It depends on a sample and an X-ray excitation source interacting ( Figure 5). EDS can be used to identify the chemical elements present in a sample and quantify their relative abundance (qualitative and quantitative analysis  25.86% in Ag/cellulose nanocomposites, It appears that Ag replaced carbon atoms in Ulva/nanocellulose and formed Ag/cellulose nanocomposites (Figure 4a,b). The analysis of the TiO 2 /CNF composite using energy-dispersive X-ray spectroscopy (EDS) is mostly composed of the three elements, C, O, and Ti [68]. Energy-dispersive X-ray (EDX) analysis was used to emphasize the elemental structure of the silver nanospheres/graphene oxide composite [69]. Silver nanoparticles were well dispersed on the surface of cellulose and penetrated into the cellulose network [70].

Energy-Dispersive X-ray Measurements
An analytical method called energy-dispersive X-ray spectroscopy can be used to determine the relative abundance of different elements in a given sample. It depends on a sample and an X-ray excitation source interacting ( Figure 5). EDS can be used to identify the chemical elements present in a sample and quantify their relative abundance (qualitative and quantitative analysis  25.86% in Ag/cellulose nanocomposites, It appears that Ag replaced carbon atoms in Ulva/nanocellulose and formed Ag/cellulose nanocomposites (Figure 4a,b). The analysis of the TiO2/CNF composite using energy-dispersive X-ray spectroscopy (EDS) is mostly composed of the three elements, C, O, and Ti [68]. Energy-dispersive X-ray (EDX) analysis was used to emphasize the elemental structure of the silver nanospheres/graphene oxide composite [69]. Silver nanoparticles were well dispersed on the surface of cellulose and penetrated into the cellulose network [70].

X-ray Diffraction
The intensity and shape of the peaks in the XRD patterns are affected by crystal size and crystalline shape. As can be seen, the XRD pattern of nanocellulose and Ulva/Ag/cellulose nanocomposites derived from U. lactuca indicate the crystal structure. The XRD diffraction patterns of nanocellulose derived from U. lactuca were recorded at 2θ, 10.  (Figure 6a and Table 2). The major crystalline peak was obtained at 2θ (31.6 • ) with an intensity of 100% and crystalline size of 27.83, which confirmed that the nanocellulose obtained is nanocrystalline. The peaks at 2θ, 21.9, 22.9, and 45.5 are the broad bands and denote the amorphous nanocellulose. The conventional two-phase cellulose model illustrates cellulose chains as comprising both crystalline (ordered) and amorphous (less ordered) regions [71]. The methods of cellulose synthesis were affected in amorphous and crystalline regions in cellulose nanofibril (CNF) [22]. The XRD peaks of CNCs at 15 • , 16.  Table 2b). When the diffraction peak is quite sharp, which indicates that the silver has good crystallization performance [73] The diffraction peaks at 38.

X-ray Diffraction
The intensity and shape of the peaks in the XRD patterns are affected by crystal size and crystalline shape. As can be seen, the XRD pattern of nanocellulose and Ulva/Ag/cellulose nanocomposites derived from U. lactuca indicate the crystal structure. The XRD diffraction patterns of nanocellulose derived from U. lactuca were recorded at 2θ, 10.  (Figure 6a and Table 2). The major crystalline peak was obtained at 2θ (31.6°) with an intensity of 100% and crystalline size of 27.83, which confirmed that the nanocellulose obtained is nanocrystalline. The peaks at 2θ, 21.9, 22.9, and 45.5 are the broad bands and denote the amorphous nanocellulose. The conventional two-phase cellulose model illus-   Figure  6b and Table 2b). When the diffraction peak is quite sharp, which indicates that the silver has good crystallization performance [73] The

Zeta Potential Analysis
From the zeta potential results presented in Figure 7a,b, it is noticeable that the surface charge of the nanocellulose derived from U. lactuca has a negative charge of −0.217 mV, and Ulva/Ag cellulose nanocomposite is−16.4 mV. The results demonstrate that Ulva/Ag cellulose nanocomposites are more negative than Ulva/nanocellulose. Abo-Elmagd et al. [76] stated that charges and strong resistive forces between the particles prevent aggregation and keep the nanoparticles in the medium stable. The zeta potential value of AgNPs photosynthesized by Oscillatoria limnetica was −27.4 [77]. The negative charge of nanoparticles revealed the repulsion among the nanoparticles and superior constancy. In agreement with this study, the Au/cellulose nanocomposite biofabricated by green alga Chlorella vulgaris is negative (−13.6 mV) [78]. The difference in zeta potential values of these results and other research may be due to the zeta potential of cellulose nanocrystal (CNC) aqueous dispersions, as was the function of solution conditions, including changing pH and different electrolyte identities and concentrations [79]. Figure 8 displays the DSC thermogram of Ulva/Ag/cellulose nanocomposites blended with fluoride, the temperature ranged from 20 • C to 600 • C. The glass transition temperature (Tg) 117.237 • C, was attained. The exothermic exhibited in the range of 148.648 and 169.788 • C may be due to the result of water evaporation. Composites exhibited initial decomposition around 100 • C, which could be due to the result of water evaporation [80]. According to the DSC curve, drug dehydration occurred between 50 and 121.8 • C [81].

Differential Scanning Calorimetry (DSC)
In the second exothermic peak, ranging from 225 and 317.12 • C, thermal decomposition of cellulose nanocomposites was exhibited. The results are in agreement with Peter and Chrebet's [82] reported cellulose decomposition beginning at temperatures of 250-260 • C. The results demonstrated the presence of exothermic peak during the heating scan of differential scanning calorimeter (DSC) analysis, called the cold crystallization peaks [83]. −0.217 mV, and Ulva/Ag cellulose nanocomposite is−16.4 mV. The results demonstrate that Ulva/Ag cellulose nanocomposites are more negative than Ulva/nanocellulose. Abo-Elmagd et al. [76] stated that charges and strong resistive forces between the particles prevent aggregation and keep the nanoparticles in the medium stable. The zeta potential value of AgNPs photosynthesized by Oscillatoria limnetica was −27.4 [77]. The negative charge of nanoparticles revealed the repulsion among the nanoparticles and superior constancy. In agreement with this study, the Au/cellulose nanocomposite biofabricated by green alga Chlorella vulgaris is negative (−13.6 mV) [78]. The difference in zeta potential values of these results and other research may be due to the zeta potential of cellulose nanocrystal (CNC) aqueous dispersions, as was the function of solution conditions, including changing pH and different electrolyte identities and concentrations [79].

Antimicrobial Activity
The agar-well diffusion technique was used to investigate the antibacterial activities of hybrids made of AgNPs, Ulva/nanocellulose, and Ulva/Ag/cellulose nanocomposite at different concentrations of nanocellulose (0.1, 0.2, 0.4, and 0.8 g) against both S. mutans ATCC 25175 and L. acidophilus CH-2.
Results obtained from the clear zone (mm) around the two bacterial types revealed that all examined nanoparticles have antibacterial activity on selected indicator organisms, however, Ulva/nanocellulose showed low antibacterial activity. Both AgNPs and Ulva/Ag/cellulose nanocomposite showed greater inhibitory zone diameter than that obtained by Ulva/nanocellulose. Furthermore, no significant differences in inhibition zone diameter were observed when different cellulose concentrations were applied to process Ulva/Ag/cellulose nanocomposites ( Figure 9 and Table 3). The interaction and toxicity of nanoparticles are more significant with the bacterial surface due to their small size and high surface area. [23]. Results in Figure 8 and Table 3 demonstrate Ulva/nanocellulose possessed low antibacterial activities against both S. mutans ATCC 25175 and L. acidophilus CH-. Nanocellulose, by nature, does not have any antimicrobial properties and needs surface modification to make it an antimicrobial material [84]. Khulbe and Matsuura [85] recorded metal/metal oxide nanoparticles (such as Cu, Au, CuO, ZnO, Ag, TiO 2 , and MgO), silanes, chlorine, and chitosan as coupling agents that provide nanocellulose with antimicrobial properties. In a previous study, nanocellulose was modified by combining it with chitosan to inhibit the growth of Escherichia coli by 99% [86]. Oxide/cellulose nanocomposite presented afflicted antibacterial activity against Staphylococcus aureus and E. coli [27]. The AgNPs cellulose composite showed excellent antibacterial activity to prohibit bacterial infection and biofilm formation efficacy against most types of bacteria [87]. These nanoparticles have different mechanisms of action, including damaging the bacterial cell membrane by transference of Ag 0 to Ag + , as well as the obstruction of the intercellular metabolic pathways following Ag + penetration into the cell [88]. Antibacterial experimental results showed that the cellulose-silver hybrids showed excellent antimicrobial activities against E. coli (Gram-negative) and S. aureus (Gram-positive) [70]. Since they exhibit antibacterial properties, CNF/ZnO films can be utilized in biological applications [89]. The antibacterial activity of silver against K. pneumonia strains (Gram-negative bacteria) and S. aureus (Gram-positive bacteria) has been reported [90]. Silver ions change the function of the bacterial wall after binding with it [91]. El-Abd et al. [92] reported that coating biosynthesis AgNPs with acetic acid reduced the microflora in Ross broiler chicks such as Pseudomonas orizihabitain. Hamouda et al. [93] estimated that biosynthesis AgNPs using two red algae capping SDS possessed antibacterial activities against Micrococcus leutus, Kocuria varians, and Escherichia coli ATCC 8739. Silver nanoparticles biofabricated by Oscillatoria limnetica demonstrated the highest antibacterial activity against multidrug-resistant bacteria (MDR) [77]. 3.6. Differential Scanning Calorimetry (DSC) Figure 8 displays the DSC thermogram of Ulva/Ag/cellulose nanocomposites blended with fluoride, the temperature ranged from 20 °C to 600 °C. The glass transition temperature (Tg) 117.237 °C, was attained. The exothermic exhibited in the range of 148.648 and 169.788 °C may be due to the result of water evaporation. Composites exhibited initial decomposition around 100 °C, which could be due to the result of water evaporation [80]. According to the DSC curve, drug dehydration occurred between 50 and 121.8 °C [81]. In the second exothermic peak, ranging from 225 and 317.12 °C, thermal decomposition of cellulose nanocomposites was exhibited. The results are in agreement with Peter and Chrebet's [82] reported cellulose decomposition beginning at temperatures of 250-260 °C. The results demonstrated the presence of exothermic peak during the heating scan of differential scanning calorimeter (DSC) analysis, called the cold crystallization peaks [83].

Antimicrobial Activity of Nanocellulose Blended with Fluoride
The results presented in Table 4 show that fluoride had no inhibition zone present with two bacterial strains, while nanocellulose blended with fluoride was more effective than nanocellulose and Ulva/Ag/cellulose nanocomposites/fluoride in the case of S. mutans ATCC 25175. Ulva/Ag/cellulose nanocomposites/fluoride possessed more activity than nanocellulose/fluoride against L. acidophilus CH-2. Fluoridated polyethylene glycol-coated silver nanoparticles (PEG-AgNPs) can be used for antibacterial activity against S. mutans [94]. Silver nanoparticles blended with fluoride had antibacterial activity against Streptococcus mutans [95]. In comparison with sodium fluoride, silver nanofluoride was more effective in inhibiting pH lowering and adherence of S. mutans to the enamel surface [96]. Widakdo et al. [97] reported graphene oxide (GO) had antibacterial qualities against Escherichia coli, and the antibacterial level increased to 99.9% in almost all membranes with different pH values, due to the synergistic effect between AgNPs, GO, GO-AgpHx that showed a lower molecular transmission resistance and a higher antibacterial effect. Linear low-density polyethylene (LLDPE) combined with CaO and metal ion forms (LLDPE/CaO Ag, LLDPE/CaO Zn, and LLDPE/CaO Cu) were excellent and functioned as antibacterial agents against E. coli with an antibacterial rate of 99.9% [98].

Antimicrobial Activity of Nanocellulose Blended with Fluoride
The results presented in Table 4 show that fluoride had no inhibition zone present with two bacterial strains, while nanocellulose blended with fluoride was more effective than nanocellulose and Ulva/Ag/cellulose nanocomposites/fluoride in the case of S. mutans ATCC 25175. Ulva/Ag/cellulose nanocomposites/fluoride possessed more activity than nanocellulose/fluoride against L. acidophilus CH-2. Fluoridated polyethylene glycol-coated silver nanoparticles (PEG-AgNPs) can be used for antibacterial activity against S. mutans [94]. Silver nanoparticles blended with fluoride had antibacterial activity against Streptococcus mutans [95]. In comparison with sodium fluoride, silver nanofluoride was more effective in inhibiting pH lowering and adherence of S. mutans to the enamel surface [96]. Widakdo et al. [97] reported graphene oxide (GO) had antibacterial qualities against Escherichia coli, and the antibacterial level increased to 99.9% in almost all membranes with different pH values, due to the synergistic effect between AgNPs, GO, GO-AgpHx that showed a lower molecular transmission resistance and a higher antibacterial effect. Linear low-density polyethylene (LLDPE) combined with CaO and metal ion forms (LLDPE/CaO Ag, LLDPE/CaO Zn, and LLDPE/CaO Cu) were excellent and functioned as antibacterial agents against E. coli with an antibacterial rate of 99.9% [98].

Minimum Inhibitory Concentration (MIC)
The MIC values of Ulva/Ag/cellulose nanocomposites were tested against S. mutans ATCC 25175 and L. acidophilus CH-2 at concentrations ranging from 0.0018 to 0.000056 g/mL. The inhibition zone diameter values (mm) were dose dependent and gradually decreased by decreasing the concentration of Ag/Ulva/cellulose nanocomposites (Table 5 and Figure 10). The results show that the MIC of both bacteria was 0.000112 g/mL (0.112 mg/L). Hamouda et al. [92] reported that the MIC of AgNPs synthesis by marine green alga Ulva fasciata is 0.5 mg/mL for S. aureus, 1.0 mg/mL for Salmonella enterica sub sp., 0.5 mg/mL for Aeromonas hydrophila, 2 mg/mL for E. coli O157, and 1.0 mg/mL for Bacillus cereus.
There are many studies that have proved the antibacterial activities of AgNPs derived from biomaterials and Ag cellulose nanocomposites against different bacteria, as tabulated in Table 6.

Minimum Inhibitory Concentration (MIC)
The MIC values of Ulva/Ag/cellulose nanocomposites were tested against S. mutans ATCC 25175 and L. acidophilus CH-2 at concentrations ranging from 0.0018 to 0.000056 g/mL. The inhibition zone diameter values (mm) were dose dependent and gradually decreased by decreasing the concentration of Ag/Ulva/cellulose nanocomposites (Table 5 and Figure 10). The results show that the MIC of both bacteria was 0.000112 g/mL (0.112 mg/L). Hamouda et al. [92] reported that the MIC of AgNPs synthesis by marine green alga Ulva fasciata is 0.5 mg/mL for S. aureus, 1.0 mg/mL for Salmonella enterica sub sp., 0.5 mg/mL for Aeromonas hydrophila, 2 mg/mL for E. coli O157, and 1.0 mg/mL for Bacillus cereus. There are many studies that have proved the antibacterial activities of AgNPs derived from biomaterials and Ag cellulose nanocomposites against different bacteria, as tabulated in Table 6.

Conclusions
Nanocomposites have led to hurriedly increasing applications in various fields. This study investigated the effect of nanocellulose and Ulva/Ag/cellulose nanocomposites derived from marine alga Ulva lactuca against S. mutans ATCC 25175 and L. acidophilus CH-2. Ulva/nanocellulose and Ulva/Ag/cellulose nanocomposites were characterized by TEM, SEM, zeta potential, EDX, XRD, and FT-IR. SEM confirmed that nanocellulose comprises filaments, and Ulva/Ag cellulose nanocomposites have fibrous surfaces. TEM images showed that the diameter of Ulva/nanocellulose ranged from 17 to 28 nm, while the diameter size of Ulva/Ag cellulose nanocomposites was 18.59 to 38.11. XRD confirmed that both Ulva/nanocellulose and Ulva/Ag cellulose nanocomposites were crystalline. Zeta potential indicated that Ulva/Ag cellulose nanocomposites were more negative in charge than Ulva/nanocellulose. Ulva/nanocellulose and Ulva/Ag cellulose nanocomposites have antibacterial activity against S. mutans and L. acidophilus. Data analysis revealed that Ulva/Ag/cellulose nanocomposites are more effective than Ulva/nanocellulose, and Ulva/Ag/cellulose nanocomposites blended with fluoride had more antibacterial activity than those not blended with fluoride. The antibacterial activities showed nonsignificant differences based on using different cellulose concentrations. Based on the data obtained, it is possible to deduce that Ulva/Ag/cellulose nanocomposites have the potential to be an economical and safe nanocomposite product from a natural source with antibacterial components that might be employed against bacteria that cause dental decay, blended to toothpaste, and add to tooth filler.

Data Availability Statement:
The datasets spent and/or analyzed during this study are available from the corresponding author upon reasonable request.